1. Field of the Invention
The present invention relates to replication of damaged DNA and to mutagenesis of DNA by highly mutagenic replication.
2. Description of the Related Art
Genomic DNA is continuously subjected to damage by internal and external agents, such as reactive oxygen species or sunlight, and by spontaneous decay (e.g., depurination). The DNA lesions produced interfere with replication and with gene expression and they must be removed by DNA repair enzymes in order to enable proper function of DNA. When unrepaired lesions are replicated, they give rise to mutations due to their miscoding potential (Friedberg et al., 1995). This is of major interest from the human disease standpoint, since the formation of mutations in critical target genes (oncogenes and tumor suppressor genes) leads to cancer. It has been estimated that most human cancers are caused by unrepaired DNA lesions (Sancar, 1994).
A broad class of DNA lesions, including UV light-induced pyrimidine cyclobutyl dimers or 6-4 adducts, abasic sites, or DNA adducts produced by certain drugs, such as cisplatin, interrupt DNA replication, leading to the formation of single-stranded regions. Such structures, ssDNA (single-stranded DNA) regions carrying damaged bases (gap lesion structures), cannot be repaired by the regular excision repair pathways, because that would lead to a double-strand break, which is highly lethal. The emergency tolerance strategy adopted for such cases is to repair (fill in) the gap without removing the damaged base. This converts the single-stranded region back into a duplex structure, thus restoring DNA continuity and reducing the risk of chromosome breakage. Excision repair mechanism might then have, at a later stage, a second chance to remove the lesion (Livneh et al., 1993; Friedberg et al., 1995).
Two general mechanisms are known for filling-in of gap lesion structures. Recombinational repair relies on the homologous fully replicated sister chromatid to provide a DNA segment that is patched across the lesion. This process is fundamentally error-free and is a major repair function in E. coli (Kowalczykowski et al., 1994; Eggleston et al., 1996). The second strategy consists of filling-in of the gap lesion by a DNA polymerase. This mechanism is mutagenic because polymerases tend to incorporate incorrect nucleotides opposite DNA lesions. In E. coil, this process, which is the paradigm for genetically regulated mutagenesis, is under tight regulation by the SOS stress response and requires specific inducible proteins. Its major outcome is a dramatic increase in mutations associated with DNA damage. It was termed error-prone DNA repair, SOS repair, or SOS mutagenesis (referring to its outcome), or translesion replication (referring to its mechanism; reviewed in Livneh et al., 1993; Walker, 1995). Two suggestions were offered to explain the function of such a system in E. coli. First is the repair of DNA gaps (opposite lesions) on which recombination cannot act (e.g., overlapping daughter strand gaps). The price of this repair is an increase in mutation frequency. The second is the facilitated adaptation of cell populations to environmental stress condition, via an inducible mutagenesis mechanism (Radman, 1975, Witkin, 1976; Bridges, 1978; Echols, 1981). The latter function is particularly intriguing because it implies an active mode of evolution (Echols, 1981).
In addition to this mutagenesis process, which is targeted to DNA lesions, a mutator activity is induced under SOS conditions, which produces mutations in the apparent absence of DNA damage (untargeted mutagenesis) (Witkin, 1974; George et al., 1975; Witkin et al., 1979). Chromosomal untargeted mutagenesis requires the SOS-inducible proteins RecA, UmuD', and UmuC (Witkin, 1976; Ciesla, 1982; Fijalkowska et al., 1997), the same proteins that are required for translesion replication. In addition, it exhibits a particular mutational specificity, namely, the selective generation of transversions (Fijalkowska et al., 1997; Miller et al., 1984; Yatagai et al., 1991; Watanabe-Akanuma et al., 1997). Another pathway of untargeted mutagenesis is observed by transfecting UV-irradiated E. coli cells with unirradiated phage .lambda. (Ichikawa-Ryo et al., 1975). This phage untargeted mutagenesis requires the dinB, uvrA, and polA gene products (Maenhaut-Michel et al., 1984; Brotcorne-Lannoye et al., 1986; Caillet-Fauquet et al., 1988; Kim et al., 1997) and produces frameshift mutations (Wood et al., 1984). Recently, dinB (a homologue of umuC) was shown to encode an error-prone DNA polymerase termed pol IV, which tends to produce frameshifts (Wagner et al., 1999). The role of pol IV in E. coli cells is not clear, because dinB mutants are proficient both in untargeted and targeted SOS mutagenesis (Brotcorne-Lannoye et al., 1986; Kenyon et al., 1980).
In E. coli, the major tolerance mechanism toward unrepaired lesions is recombinational repair. In contrast, recombinational repair in mammals is less active (Friedberg et al., 1995), perhaps because of the large proportion of repetitive sequences in the mammalian genome, which increases the danger of undesired gross rearrangements. This leaves translesion replication as the major candidate for tolerance of unrepaired lesions in mammals. The scarcity of knowledge on mammalian tolerance of DNA damage underscores the importance of elucidating similar mechanisms in model organisms such as bacteria and yeast.
Based on genetic analysis, SOS mutagenesis in E. coli required DNA polymerase III (Bridges et al., 1976; Brotcorne-Lannoye et al., 1985), which is the replicative DNA polymerase, as well as three SOS-inducible proteins: RecA, UmuD', and UmuC. RecA is a multifunctional protein, known to be the major recombinase in E. coli (Roca and Cox, 1990), but its function in SOS mutagenesis is not directly related to recombination. RecA fulfills three roles in SOS mutagenesis, of which two are regulatory (Witkin, 1991): First, it activates the SOS stress response by promoting the cleavage of the LexA repressor. This induces the expression of the mutagenesis-specific proteins UmuD and UmuC. Second, it promotes the posttranslational cleavage of UmuD to UmuD', the active form in mutagenesis (Burckhardt et al., 1988; Nohmi et al., 1988; Shinagawa et al., 1988). In addition, RecA has been suggested to have a third, presumably direct, role in the mutagenic process (Dutreix et al., 1989; Sweasy et al., 1990). UmuD' and UmuC are specifically required for SOS mutagenesis (Kato and Shinoura, 1977). A pioneering study by Rajagopalan et al. (1992) indicated the UmuD' and UmuC act as bypass factors and increase translesion replication by pol III holoenzyme. However, the further utilization of that experimental system was hampered by the difficulty in obtaining purified active UmuC (Woodgate et al., 1989).
Two homologues of the E. coli umuC gene were recently found in the yeast S. cerevisiae. The REV1 gene is required for UV mutagenesis and encodes a dCMP transferase (Nelson et al., 1996). The RAD30 gene encodes DNA polymerase .eta., a translesion replication DNA polymerase which effectively and accurately bypasses cyclobutyl pyrimidine dimers, the major UV lesions (Johnson et al., 1999 and Johnson et al.; 1996). In addition yeast cells contain DNA polymerase .zeta., which is required for UV mutagenesis, but is not a homolog of umuC. It is encoded by the REV3 and REV7 genes (Nelson et al., 1996). Human cells contain 4 proteins which belong to this superfamily: DNA polymerase .eta. is encoded by the XP-V (hRAD30A) gene (Masutani et al., 1999 and Johnson et al., 1999). This protein is defective in the genetic disease Xeroderma Pigmentosum Variant, which causes sunlight sensitivity, and predisposition to skin cancer. The function of two other homologues, DNA polymerase .iota., encoded by hRAD30B (McDonald et al., 1999 and Tissier et al., 2000), and DNA polymerase .theta., encoded by hDINB1 (Gerlach et al., 1999 and Johnson et al., 2000); also termed DNA polymerase .kappa., (Ohashi et al., 2000), is still unknown. Human cells contain also the REV1 gene, which encodes a dCMP transferase (Lin et al., 1999 and Gibbs et al., 2000), and a homologue of the yeast REV3 gene, which were shown to be required for UV mutagenesis in human cells (Gibbs et al., 1998).
An interesting group of umuC and umuD homologues contains genes residing on native conjugative plasmids. These plasmids have a broad host range specificity, and they often carry multiple antibiotics resistance genes (Woodgate et al., 1992). Their existence in human pathogenic bacteria may account, in part, for the growing problem of antibiotics resistance among bacterial pathogens (Davies, 1994; Dennesen et al., 1998 and Swartz, 1994). The most extensively studied of these is the mucAB operon, carried on plasmid pKM101 (Perry et al., 1982), which is a natural variant of plasmid R46. Plasmid pKM101 was introduced into the Salmonella strains used in the Ames test for mutagens, where it increased the sensitivity of the assay via mucAB-mediated mutagenesis (McCann et al., 1975). Other known plasmidic umuDC homologues include, impCAB (Lodwick et al., 1990), Sam AB (Nohmi et al., 1991), and rum AB (Kulaeva et al., 1995). The laboratory of the present inventors have previously overproduced MucA, MucA' and MucB, and showed that MucA' forms a homodimer, and that MucB is a ssDNA-binding protein (Sarov-Blat et al., 1998). In addition, the laboratory of the present inventors found that MucB interacts with a SSB-coated ssDNA, causing a major conformational change, but without causing massive dissociation of SSB from the DNA (Sarov-Blat et al., 1998).
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